Chapter 7 The Plasma Protein

Tu Jiancheng

contents

1. Concept of Plasma Protein

2. Total Protein: Acute-phase Reactants Individual Proteins and Disease States

3. Methods of Protein Analysis

1. Concept of Plasma Protein

The Plasma Proteins Are A Diverse Group of Molecules ThatPerform A Variety Of Functions

* Need to Be Distinguished from Proteins That Occur Only Incidentally in The Blood.

* Often Classified Based on Their Electrophoretic Mobility

* Most of Them are Synthesized in the liver

Densitometric scan of a normal serum protein electrophoresis pattern showing the relative position of the albumin,α1,α2,β and γregions

Selected Majora and Minor Plasma Proteins (TABLE 7-2 )

Albumin zone Albumin Prealbumin

α1 Zone

α1 –Antitrypsin

High-density lipoprotein (α-lipoproteins) α1-Antichymotrypsin

Orosomucoid α-Fetoprotein

α2 Zone

α2 –Macroglobulin

Haptoglobin Ceruloplasmin Gc-Globulin

βZone Low-density lipoprotein (β-lipoproteins) Transferrin C3 β2-Microglobulin

Hemopexin Fibrinogen (may be inγZone)

γZone Immunoglobulins C-Reactive protein Fibrinogen Lysozyme

a In boldface

Plasma protein abnormalities arise from one or more of the following:

* Congenital abnormalities affecting a specific protein

* Acquired abnormaltities affecting a specific protein

* Alterations affecting multiple proteins reflecting variations in the physiologic state

* Alterations affecting multiple proteins secondary to disease

2. Total Protein

Factors Which Affect Protein Concentration: * Fluid Balance * Changes in Synthesis or Catabolism

* Protein losses.

* Prolonged Tourniquet Application during Venipuncture Increase the Total Protein by as much as 5g/L.

* Recumbent Subjects 10% lower than in Ambulatory Ones * Normal Diurnal Variation of 4 g/L

* Seasonal Fluctuation: Peak in Nov. and Lowest in June. * Exercise increases the serum protein concentration by as much as 10%, but this effect is transient.

Preanalytic Errors Which May Alter the Serum Protein value

* The total serum protein includes both albumin (60%) and the globulins (40%)

* The total protein concentration may be altered by changes in fluid balance

* Changes in the amounts of plasma proteins may result from alterations in synthesis or catabolism or from protein losses

Hypoproteinemia

* decreased protein synthesis

* malnutrition

* chronic liver disease

Nephritic syndrome

* the serum albumin level may fall to less than 5 g/L

ACUTE-PHASE REACTANTS(APRs)

A Group of Proteins Whose Plasma Concentration Changes In Response To A Variety of Inflammatory Sates Including: * Infection * Surgery * Trauma* Myocardial Infarction * Malignancy * Any Condition Associated with Tissue Necrosis

Cytokines

* Major Stimulator: Interleukin-6 (IL-6) is the

* Other Cytokins Which Upreugulate APRs: Interleukin-1β(IL-1β), Tumor Necrosis Factor-α(TNF-α) Interferon Gamma (IFN-γ) Transforming growth factorβ(TGF-β)

Regulation of APRs

Positive Negative

α1-Antitrypsin Albumin

α1-Antichymotrypsin Prealbumin

Haptoglobin Retinol binding protein

Ceruloplasmin Transferrin (rises in late acute phase)

Fibrinogen

C3

C-Reactive protein

Hemopexin

Serum amyloid A protein

Major Acute Phase Reactants (TABLE 7-3 )

* Measurement of APRs, especially C-reactive protein (CRP), may be useful to detect and follow patients with acute inflammatory disorders.

The changes in plasma proteins seen

during the acute-phase response

potentially serve a variety of functions

* Positive APRs: AAT,α1-Antichymotrypsin.

* Protect against reactive oxygen species: haptoglobin, hemopexin

Individual Proteins and Disease States

PrealbuminAlbuminα1-Antitrypsinα2-Macroglobulin CeruloplasminHaptoglobinTransferrinβ-Lipoproteinβ2-MicroglobulinC-Reactive Protein

* Prealbumin is a tryptophan-rich tetrameric non-glycosylated protein named for its anodal migration relative to albumin on protein electrophoresis.

Prealbumin(1)

[MW]: 55 kD [Synthesized]: in Liver[Synonyms]: Transthyretin and Thyroid-Binding Prealbumin. [Function]: Carrier Protein for Thyroid Hormone and for vitamin A (with Retinal Binding Protein) [Half-Lives]: 1.9 days. [Reference Range]: 195 - 358 mg/L

Prealbumin(2)

* Prealbumin levels reflect hepatic synthesis and can serve as an index of liver function

* Prealbumin is a compact molecule that can cross the blood-brain barrier and may also be synthesized by cells of the choroids plexus.

* Prealbumin can be measured by a variety of immunoassays, including radial immunodiffusion and nephelometry.

Albumin(1)

The most abundant protein in plasma, comprising approximately 60% of the total protein concentration.

[MW] 66.3kD, Most Abundant protein in Plasma, [Synthesized] almost exclusively by the liver,[Half-life] 19 days.[Functions]1. 80% of the plasma COP. 2. Amino Acid Source to a Variety of Cells 3. Major Transport Protein 4. Lipid Metabolism [Reference Range] 31 to 43g/L.

Albumin (2)

[measure]

Electrophorectic

Immunochemical

dye-binding techniques

bromocresol purple

Bromocresol green

Albumin(3)

α1-Antitrypsin (AAT)(1)

One of the major plasma proteins, comprising nearly 90% of the α1 globulin region on SPE.

Deficiency of AAT has been associated with pulmonary emphysema and hepatic cirrhosis.

[MW]: 52kD, Major Plasma Proteins, [Function]: a Serine Proteinase Inhibitors(Serpins )[Synthesizied]: in Liver. Seen in Plasma, Tears, Lymph, Bile, Semen, and Amniotic [Acquired decreases]:•Protein-losing syndromes, •malnutrition, •severe liver damage, •respiratory diseases: neonatal respiratory distress syndrome [Congenital Deficiency]:•Pulmonary Emphysema and Hepatic Cirrhosis[Reference Range]: 900~1500mg/L

α1-Antitrypsin (AAT)(2)

* Genes encoding the AAT protein comprise an autosomal allelic system containing at least 75 codominant genes inherited on a single locus called Pi for proteinase inhibitor.

α1-Antitrypsin (AAT)(3)

• The PiZ allele is the most common variant associated with AAT deficiency.

PiZZ : 10% to 15% (normal)

PiMZ: 60% (normal)

PiSS and PiMS: 63% and 83%(normal)

α1-Antitrypsin (AAT)(4)

* Consists of four identical subunits and is one of the largest serum proteins

* Can inhibit Several endopeptidases

* Can bind numerous growth factors, hormones, and cytokines

α2-Macroglobulin(AMG)(1)

[MW] 720 kD, Largest Serum Protein [Synthesized] in Liver [Half Life] 5 Day in Serum

[Function] 1. Defense Against Proteolytic Enzymes (Irreversibly Binds Proteinases ) 2. Transport Protein? (Binds to Growth Factors, Hormones, and Cytokines)

[Reference Range]125 to 215 mg/dL

α2-Macroglobulin(AMG)(2)

Ceruloplasmin(CER)(1)

* A glycoprotein synthesized by the liver as a single polypeptide chain to which six to eight copper atoms are attached.

* The pure protein is blue because of its high copper content. Increased levels may impart a green tinge to plasma samples.

[MW] 132kD, a Glycoprotein, Synthesized by the Liver, not Normally Visible on Routine Gels[Clinical Significants]: Diagnosis of Wilson’s Disease (Hepatolenticular Degenetation)

[Reference Range]: 27 to 50 mg/dL Highest in young children and somewhat Lower in both Infants and Adults. Less than 10 mg/dL are Suggestive of Wilson’s Disease,

Ceruloplasmin(CER)(2)

Haptoglobin(1)

* A glycoprotein synthesized by the liver that migrates as an α2 globulin.

* In addition to its well-known function in the binding of free hemoglobin, this APR might also play an important role in the control of local inflammation.

Glycoprotein [Synthesized] in the liver [Migrates] as α2 globulin [Function] Binding of Free Hemoglobin, Control of Local Inflammation.[Clinical Significatns] Decreased Associated with Intravascular Hemolysis, (Hemolytic Anemias, Hemoglobin-opathies, Hemolytic Transfusion Reactions, Extensive Burns, Malaria, Disseminated Intravascular Coagulation, and Exercise-Induced Hemolysis). [Reference range] 16 to 199 mg/dL.

Haptoglobin(2)

Transferrin

[MW]: 80kD, a Glycoprotein[Synthesized] by Liver(most), reticuloendothelial system, the gonads, and the submaxillary gland [Function] Transport of Iron from Intestine (or Hemoglobin catabolism) to Red Cell[Reference Range] 191 to 365 mg/dL (1.91 to 3.65g/L).

β-Lipoprotein(1)

* Lipoproteins constitute a family of molecules composed of lipids and proteins whose function is to transport cholesterol, triglycerides, and phospholipids in the blood.

β-Lipoprotein(2)

[Components] Chylomicrons(CM), [Very Low Density Lipoproteins (VLDL) [Low-Density Lipoproteins (LDL) [High-Density Lipoproteins (HDL).

β2-Microglobulin(1)

* A small protein (MW 11,818) comprising the common light chain of the class I major histocompatibility complex antigen on the surfaces of all nucleated cells.

[MW] ~11,818 Dalton, Noncovalently Linked to the human lymphocyte antigen (HLA) heavy chain. [Distribution] Plasma, Urine, and CSF[Function] Necessary for Insertion of the HLA into Cell Membrane, and Stabilize the HLA heavy chain. It Regulation of Some Lymphocyte Functions.[The normal Reference Range] Serum 2.8mg/L Urine 0.2mg/L

β2-Microglobulin(2)

* CRP was first identified in 1930 as a substance present in the sera of patients with pneumococcal pneumonia that could bind to C-polysaccharide isolated from Streptococcus pneumoniae, producing a flocculation reaction.

C-Reactive Protein(CRP)(1)

[MW] 118,000 Dal [Physiology] Elevated in a Variety of Acute Inflammatory siseases. [Half Life] 19 hours. [Synthesized] by the liver and released into the plasma. Peripheral(Small Amount)[Function] Recognition of Microbial Organisms An immunomodulator in Host Defense. Recognition of Necrotic Tissues. [Reference Range] Less than 0.8mg/dL..

C-Reactive Protein(CRP)(2)

MTEHODS OF PROTEIN ANALYSIS

* Measurement of Total Protein

* Serum Protein Electrophoresis

* Other methods

Measurement of Total Protein

☆Kjeldahl procedure

☆ biuret method

☆ Lowry method

☆ dye-binding procedures

☆ turbidimetric method

☆ nephelometric method

Serum Protein Electrophoresis

* Principles of Electrophoresis

* Agarose Gel Electrophoresis

* Capillary Elecrophoresis

Principles of Electrophoresis

＊ support media ＊ buffer ions ＊ pH ＊ the size and shape of the molecule ＊ the strength of the electric field ＊ the temperature ＊ the effects of convection and diffusion ＊ the ionic and pore properties of the ＊ electrophoresis medium

Typical serum protein Electrophoresis Reference Pattern (TABLE 7-4)

ZonePercentage of Total Protein (mean)

Albumin 45.8-68.2 (57.0)

α1 2.1-6.1 (4.1)

α2 8.8-18.0 (13.4)

β 7.8-13.0 (10.4)

γ 7.8-22.6 (15.2)

Representative patterns of serum protein seen on serum protein electrophoresis

Capillary Elecrophoresis

* Electrophoretic separation of analytes is carried out in fused-silica capillaries with an inner diameter of 50 μm (range, 20 to 200 μm), an outer diameter of 375 μm, and an effective length of approximately 50 cm (range, 7 to 100 cm).

* Ion Exchange Chromatogrphy

* Affinity Chromatogrphy

* Isoelectric Focusing

Other Methods